1. Field of the Invention
This invention relates to antiparasitic agents and more particularly to natural and non-natural avermectins wherein one or both of the 3'- and 3"-positions of the oleandrose disaccharide moiety is substituted by an ethoxy rather than a methoxy group; and to a process for their preparation.
2. Description of the Prior Art
U.S. Pat. Nos. 4,310,519 and 4,429,042 describe the avermectins, a complex of related agents having potent antiparasitic activity, and their production by aerobic fermentation of strains of Streptomyces avermitilis; namely, S. avermitilis ATCC Nos. 31267, 31271 and 31272. The last two strains cited represent a frozen vial and a lyophilized tube, respectively of a culture obtained by ultraviolet irradiation of S. avermitilis ATCC 31267.
EP 214,731, published Mar. 18, 1987, the counter-part of U.S. patent application Ser. No. 886,867, filed Jul. 16, 1986, discloses a number of compounds (referred to herein as non-natural avermectins) related to the natural or known avermectins but having a novel substituent group at the 25-position, and a process for their preparation by fermentation of an avermectin producing organism in the presence of certain specified carboxylic acids, or derivatives or precursors thereof. The S. avermitilis organisms used to produce the said novel C-25 substituted avermectins are S. avermitilis ATCC 31267, 31271, 31272 and NCIB 12121. The latter organism, described in EP 214,731, is derived from S. avermitilis ATCC 31271. It gives improved yields of the novel C-25 substituted avermectins when it is cultured in a semi-defined medium. Each of ATCC 31267, 31271, 31272 and NCIB 12121 may also produce, in addition to the novel C-25 substituted derivative, varying amounts of the known, or natural, avermectins wherein the 25-substituent is isopropyl or (S)-sec-butyl (1-methylpropyl).
U.S. patent application Ser. No. 6512, filed Jan. 23, 1987, discloses S. avermitilis mutants ATCC 53567 and 53568, each of which lacks branched-chain 2-oxo acid dehydrogenase activity, and their use to produce non-natural avermectins. Also disclosed is the use of O-methyltransferase inhibitors such as sinefungin, S-adenosylethionine and S-adenosylhomocysteine in fermentations of said organisms to produce increased amounts of B avermectins, including those lacking methyl groups on the disaccharide moiety.
The carbon skeleton of the avermectins (depicted in formula (I) below) is derived from acetates and propionates and the C-25 substituent of natural avermectins from L-isoleucine (R=(S)-sec-butyl) or L-valine (R=isopropyl) [Fisher and Mrozik, "Macrolide Antibiotics", Academic Press (1984) Ch. 14].
By "known" or "natural" avermectins is meant those avermectins are produced by S. avermitilis ATCC 31267, ATCC 31271 and ATCC 31272 wherein the 25-position substituent is either isopropyl or (S)-sec-butyl(1-methylpropyl). Avermectins wherein the 25-position substituent is other than isopropyl or sec-butyl (S-form) are referred to herein as novel or non-natural avermectins.
The strains of S. avermitilis cited in the above-mentioned U.S. Pat. Nos. produce a class of substances described generically therein as C-076. The class comprises eight distinct but closely related compounds described as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b. The "a" series of compounds refers to the natural avermectins wherein the 25-substituent is (S)- sec-butyl and the "b" series to those wherein the 25-substituent is isopropyl. The designations "A" and "B" refer to avermectins wherein the 5-substituent is methoxy or hydroxy, respectively. Lastly, the numeral "1" refers to avermectins wherein a double bond is present at the 22-23 position; and numeral "2" to avermectins having a hydrogen at the 22-position and hydroxy at the 23 position.
In this application no such identifiers are used as regards the 25-substituent of the non-natural avermectins. Identifiers A1, A2, B1 and B2 have been retained to refer to non-natural avermectins having the structural features corresponding to those of the natural avermectins as noted above.
U.S. Pat. No. 4,378,353 describes C-076 related compounds and their preparation by cultivation of MA-5218, a mutant strain of S. avermitilis ATCC 31272, obtained therefrom by ultraviolet irradiation. The mutant is identified as ATCC 31780. The C-076 related compounds produced by said mutant lack the C-076 furan ring. Additionally, in certain of the compounds reported, one or both of the oleandrose sugar moieties have been cleaved while in others the 5-position group was oxidized to a keto group.
Three classes of O-methyltransferase mutants of S. avermitilis that produce avermectins lacking O-methyl groups have been reported by Ruby et al., 6th International Symposium on the "Biology of Actinomycetes", Debrecen, Hungary, Aug. 26-30 (1985) and by Schulman et al, Antimicrobial Agents and Chemotherapy 31, 744-7 (1987). The first class produces primarily B avermectins due to their inability to methylate the C-5 hydroxyl of the macrocyclic lactone ring. The second class produces 3'-O, 3"-O-bis-demethylavermectins (avermectins lacking the O-methyl substituent at the 3 position of both oleandrose monosaccharide residues), and which are referred to as demethylavermectins. The third class is unable to methylate at any position.
Schulman et al., Fed. Proc. 44, 931 (1985) disclose increased production of B avermectins by fermenting S. avermitilis in the presence of substances such as sinefungin, S-adenosylethionine and S-adenosylhomocysteine which inhibit the enzyme avermectin B-O-methyltransferase which catalyzes the transfer of the methyl of S-adenosylmethionine to the C-5 hydroxy group. Streptomyces avermitilis mutants which lack O-methyltransferase activity and produce increased amounts of avermectin B components are also disclosed and referred to by Schulman et al. in Antimicrobial Agents and Chemotherapy 29, 620-624 (1986).
S. avermitilis Agly-1, a mutant strain which produces virtually only avermectin aglycones A1a and A2a is reported by Schulman et al. J. Antibiot. 38(11), 1494-1498 (1985). Also reported is the fermentation of S. avermitilis Agly-1 in the presence of sinefungin which caused increased production of avermectin aglycone B components. Likewise, S. avermitilis O8, a high producing strain for avermectins, when fermented in the presence of sinefungin as inhibitor of O-methyl transferases, resulted in production of avermectins lacking O-methyl groups on the aglycone at C-5 and in the oleandrose disaccharide moiety.
Schulman et al., Antimicrob. Agents Chemother. 31, 744-7 (1987) speculate that although not yet demonstrated, it is highly probable that since methylation of the 3'- and 3"-positions of the disaccharide moiety of avermectins is inhibited by sinefungin, S-adenosylmethionine is involved in methylation of the disaccharide moiety.
The natural avermectins, as noted, are produced as a complex mixture of eight distinct but closely related compounds; formula (I), R=isopropyl and (S)-sec-butyl. While they have been recovered in substantially pre form (see U.S. Pat. No. 4,429,042), the methodology is, at best, laborious.
The fermentation-produced avermectins, natural and non-natural alike, all have methoxy groups at the 3'- and 3"-positions of the disaccharide moiety. Production of avermectins, especially novel avermectins, bearing substitutents which impact upon their solubility and/or polarity and, hence, upon their physiological and biological properties is a desirable goal. A further desirable goal is the ability to produce said avermectins so as to minimize the number and complexity of the products, and by so doing to increase the purity of a chosen avermectin, and thereby to simplify separation procedures.
In spite of the disclosures regarding the use of SAE as an inhibitor of avermectin B O-methyltransferase, there is no disclosure of its value as an ethylating agent for the disaccharide moiety and no mention of the formation of ethylated avermectins in the presence of SAE. The use of SAE as an avermectin ethylating agent was unintended and unappreciated, and the production of ethylated avermectins was not recognized by prior investigators.